The present invention concerns a device for culturing a mass of organic cells and studying the electrophysiological activity of the cells, in which the cells are placed on at least one porous membrane, the lower surface of which contacts a liquid nutrient, said device comprising at least one electrode in contact with said mass of organic cells.
The present invention also concerns a membrane made of porous synthetic material and the use of said membrane to form a hemato-encephalic barrier.
Various devices currently exist for measuring the electrophysiological activity of a mass of organic cells.
In particular, such a device is described in French Patent Application No. FR-A-2 733 055. This document describes a device for keeping tissue explants alive and for the monitoring and continuous analysis of the electrophysiological and biochemical activity of the tissue being studied. The device is formed of two half cards which form the upper and lower half, respectively, of the interface and which are assembled to form one card that can be inserted inside an electronic case designed specifically for this purpose.
This device gives satisfactory results, but it also has a certain number of disadvantages. The electrodes are actually forced toward the cells until they contact the cells. This injures a certain number of cells, shortening their life span. After a certain length of time, the electrodes and the cells become tightly attached to each other. When it is necessary to remove the cells, for example, to perform microscopic analysis, some of them remain attached to the electrodes, which destroys the structure of the cell mass and renders them useless.
In this device the cells are nourished from below and the electrodes are placed upon the cell mass. Thus, these electrodes block the tissues from view. It is important to be able to see the cells in order to determine how the tissues are organized and which electrodes should be used. Another reason is for controlling tissue survival.
In addition, the fact that the electrodes are placed on the cells prevents intervention on the tissues being analyzed. Furthermore, this means that the electrodes must possess a relatively high degree of mechanical resistance, since they are formed of copper tracks that do not rest on a substrate. Additionally, because the card is formed of two half-cards, a large number of pieces must be manufactured and assembled.
Since the electrodes are located between the two half-cards, one end of the electrode must be placed in an area that is accessible through an electrical connector. Since each card is a single-use card, the costs increase due to the number of pieces required and the complexity of the device.
Other devices with glass substrates have also been developed. In these devices, biological tissues must be attached in order to adhere to the substrate. Since the substrate is not porous, the device must be placed in a moving apparatus that successively submerges and lifts the tissue to allow respiration. This device is heavy and does not allow long-term survival once movement has stopped. Furthermore, it is difficult to make several substrates simultaneously on one plate. Making these substrates is an especially long and expensive process.
The present invention eliminates these disadvantages with an economical device made of a small number of pieces for electrophysiological and/or microscopic cell analysis with no cell destruction.
These goals are achieved using a device such as the device described in the preamble, characterized in that the electrodes are located on the porous membrane.
According to a preferred embodiment, the device comprises a network of electrodes.
Each electrode advantageously comprises an analysis zone which can be placed in contact with the cell mass and a measurement zone which can be placed in contact with an apparatus that either generates an electrical signal and/or measures an electrical signal.
The porous membrane is preferably maintained on a rigid support.
Said rigid support advantageously contains a liquid nutrient supply chamber which communicates with a liquid nutrient inlet duct and a liquid nutrient outlet duct and which has an opening communicating with said porous membrane.
According to a preferred embodiment, a capsule which maintains the organic cells in a controlled environment surmounts the porous membrane. This capsule may comprise a gas injection duct and a gas exhaust duct.
According to a particular embodiment of the invention, the electrode measurement zones in the electrode network are arranged in a circle.
The electrode network advantageously includes a position indexing means.
According to a preferred embodiment, the porous membrane is transparent.
The objectives of the invention are also achieved by a membrane such as the membrane described in the preamble, characterized in that it comprises at least one electrode placed upon said membrane.
According to a preferred embodiment, the membrane comprises a network of electrodes placed on said membrane.
According to a preferred embodiment, each electrode comprises at least one analysis zone, one measurement zone, and one connection zone.
Finally, the objectives of the invention are also achieved by a method of making a hemato-encephalic barrier model from a membrane such as that described above, said method characterized by the steps of treating the porous membrane so the endothelial cells will adhere to it, placing the endothelial cells on a surface of the porous membrane that does not have an electrode, cultivating said endothelial cells until a layer of cells has formed, and placing a slice of organotypical culture on the other surface of the porous membrane which has at least one electrode.